The neural crest is a heterogeneous collection of progenitors that migrates out of the dorsal neural tube in early to mid-gestation, depending on the species. These cells migrate throughout the embryo and give rise to the peripheral nervous system (PNS) including sensory, sympathetic, parasympathetic, and enteric ganglia, as well as peripheral nerves. The neural crest includes neural crest stem cells (NCSCs) as well as restricted progenitors. NCSCs self-renew and undergo multilineage differentiation to form neurons, glia, and mesectodermal derivatives in culture and in vivo. Thus, to understand PNS development it is critical to understand the mechanisms that regulate the self-renewal and differentiation of NCSCs. We have developed techniques to identify uncultured NCSCs based on marker expression, to isolate these cells by flow-cytometry, to assay their self-renewal and multilineage differentiation in clonal culture, and to study their migration and differentiation in vivo. Although these techniques originally required the use of rat NCSCs, during the current funding period we developed similar techniques for studying mouse NCSCs, making it possible to apply mouse genetics to study the regulation of NCSC function. To identify new mechanisms that regulate NCSC function and PNS development, we performed gene expression profiling to identify transcripts that were significantly more highly expressed in fetal gut NCSCs as compared to whole fetal RNA. We discovered that Leucine-rich glioma inactivated gene-4 (Lgi4), which encodes a secreted protein, was much more highly expressed in NCSCs. No member of the Lgi gene family has yet been characterized in gene-targeted mice;however, Lgi4 is mutated in the spontaneously occurring claw paw mice that exhibit impaired peripheral nerve myelination. Nonetheless, Lgi4 is not known to play any role in the regulation of NCSC function or in PNS development outside of peripheral nerves. To test whether Lgi4 plays a broader role in PNS development we have generated a gene-targeted Lgi4LacZ allele that appears to give a complete loss of Lgi4 function, and a more severe phenotype than observed in claw paw mice. By studying Lgi4 expression and function using Lgi4LacZ mice, our preliminary data suggest that Lgi4 plays a much more extensive role in PNS development than could be appreciated from the claw paw mutation. We hypothesize that Lgi4 secretion by NCSCs and other neural crest cells promotes the expansion of undifferentiated NCSCs during fetal development (Aim 1) and that Lgi4 is later required for normal gliogenesis in developing peripheral nerves (Aim 2) as well as in other regions of the developing PNS (Aim 3). We further hypothesize that Lgi4 has these effects by binding and activating ADAM22 receptor signaling in neural crest cells (Aim 4). These studies have the potential to identify a novel mechanism that regulates the expansion of NCSCs as well as gliogenesis throughout the developing PNS. PUBLIC HEALTH RELEVANCE: The purpose of this project is to study the mechanisms that regulate peripheral nervous system (PNS) development. We hypothesize that we have identified a protein that regulates the expansion of neural crest stem cells and their differentiation into glial cells in the developing PNS. By better understanding these mechanisms we will gain new insights into how the PNS forms and what goes wrong in the context of disease.